Novobiocin analogues with second-generation noviose surrogates

Hsp90 is a promising therapeutic target for the treatment of cancer. Novobiocin is the first Hsp90 C-terminal inhibitor ever identified and recent structure-activity relationship studies on the noviose sugar identified several commercially available amines as suitable surrogates. In an effort to further understand this region of the molecule, analogues containing various N′-amino substituents were prepared and evaluated against two breast cancer cell lines for determination of their efficacy. Compound 37j manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation at mid nano-molar concentrations

A practical method for predicting frequent use of emergency department care using routinely available electronic registration data.

Accurately predicting future frequent emergency department (ED) utilization can support a case management approach and ultimately reduce health care costs. This study assesses the feasibility of using routinely collected registration data to predict future frequent ED visits

Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site

Novologues Containing a Benzamide Side Chain Manifest Anti-proliferative Activity Against Two Breast Cancer Cell Lines

The heat shock protein 90 (Hsp90) folding machinery is essential for the maturation of nascent polypeptides into their biologically active three-dimensional-structures and for the rematuration/clearance of misfolded proteins that form under cellular stress.1–3 As a prosurvival chaperone, Hsp90 overexpression is commonly observed in transformed cells, which is required to sustain the hostile tumor micro-environment associated with nutrient deprivation and hypoxia. Pharmacological inhibition of Hsp90 has been shown to induce the degradation of oncogenic proteins associated with all six hallmarks of cancer that rely upon Hsp90.4–8 Consequently, Hsp90 represents a highly sought after target for the treatment of cancer. In fact, 17 small molecules that bind competitively to the N-terminal ATP-binding pocket are under clinical evaluation against various cancers.9,10 However, heat shock factor 1 (HSF-1), the master regulator of the pro-survival heat shock response also binds Hsp90. Ultimately, Hsp90 N-terminal inhibition results in HSF-1 release, and upon phosphorylation, trimerizes and translocates to the nucleus wherein it binds the heat shock elements to activate the pro-survival, heat shock response (HSR). The HSR serves to expand the cellular buffering capacity and to assist in the maturation of mutated and oncogenic substrates.11 This concomitant heat shock response is detrimental to the treatment of cancer and may lead to drug resistance and tumor metastasis.12 Recent studies have demonstrated that allosteric modulation of the Hsp90 C-terminus can separate the pro-survival heat shock response from pro-apoptotic, client protein degradation.13–20 Two classes of small molecules derived from novobiocin 1, (Figure 1) the first identified Hsp90 C-terminal inhibitor, were discovered via the structure-activity relationship studies. KU-32 (2), which lacks a 4-hydroxyl, the 3’-carbamate, and contains an acetamide in lieu of a prenylated benzamide, represents a lead compound that induces the heat shock response at concentrations much lower than that needed for client protein degradation.2,21 Consequently, this class of analogues has been evaluated as neuroprotective agents to refold protein aggregates.22–24 In contrast, KU-174 (3) contains a biarylamide side chain in lieu of the acetamide, and induces Hsp90 client protein degradation without induction of the heat shock response.25–26 Therefore, this class of novobiocin analogues manifests optimal properties for the treatment of cancer, as no HSR is observed with such compounds

A C-Terminal Heat Shock Protein 90 Inhibitor Decreases Hyperglycemia-induced Oxidative Stress and Improves Mitochondrial Bioenergetics in Sensory Neurons

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in which hyperglycemia-induced mitochondrial dysfunction and enhanced oxidative stress contribute to sensory neuron pathology. KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple sensory deficits associated with the progression of DPN and protects unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically, KU-32 increased the expression of Hsp70 and this protein was critical for drug efficacy in reversing DPN. However, it remained unclear if KU-32 had a broader effect on chaperone induction and if its efficacy was linked to improving mitochondrial dysfunction. Using cultures of hyperglycemically stressed primary sensory neurons, the present study investigated whether KU-32 had an effect on the translational induction of other chaperones and improved mitochondrial oxidative stress and bioenergetics. A variation of stable isotope labeling with amino acids in cell culture called pulse SILAC (pSILAC) was used to unbiasedly assess changes in protein translation. Hyperglycemia decreased the translation of numerous mitochondrial proteins that affect superoxide levels and respiratory activity. Importantly, this correlated with a decrease in mitochondrial oxygen consumption and an increase in superoxide levels. KU-32 increased the translation of Mn superoxide dismutase and several cytosolic and mitochondrial chaperones. Consistent with these changes, KU-32 decreased mitochondrial superoxide levels and significantly enhanced respiratory activity. These data indicate that efficacy of modulating molecular chaperones in DPN may be due in part to improved neuronal mitochondrial bioenergetics and decreased oxidative stress

Induction of heat shock protein 70 (Hsp70) prevents neuregulin-induced demyelination by enhancing the proteasomal clearance of c-Jun

Modulating molecular chaperones is emerging as an attractive approach to treat neurodegenerative diseases associated with protein aggregation, DPN (diabetic peripheral neuropathy) and possibly, demyelinating neuropathies. KU-32 [N-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-yl)acetamide] is a small molecule inhibitor of Hsp90 (heat shock protein 90) and reverses sensory deficits associated with myelinated fibre dysfunction in DPN. Additionally, KU-32 prevented the loss of myelinated internodes induced by treating myelinated SC (Schwann cell)-DRG (dorsal root ganglia) sensory neuron co-cultures with NRG1 (neuregulin-1 Type 1). Since KU-32 decreased NRG1-induced demyelination in an Hsp70-dependent manner, the goal of the current study was to clarify how Hsp70 may be mechanistically linked to preventing demyelination. The activation of p42/p44 MAPK (mitogen-activated protein kinase) and induction of the transcription factor c-Jun serve as negative regulators of myelination. NRG1 activated MAPK, induced c-Jun expression and promoted a loss of myelin segments in DRG explants isolated from both WT (wild-type) and Hsp70 KO (knockout) mice. Although KU-32 did not block the activation of MAPK, it blocked c-Jun induction and protected against a loss of myelinated segments in WT mice. In contrast, KU-32 did not prevent the NRG1-dependent induction of c-Jun and loss of myelin segments in explants from Hsp70 KO mice. Overexpression of Hsp70 in myelinated DRG explants prepared from WT or Hsp70 KO mice was sufficient to block the induction of c-Jun and the loss of myelin segments induced by NRG1. Lastly, inhibiting the proteasome prevented KU-32 from decreasing c-Jun levels. Collectively, these data support that Hsp70 induction is sufficient to prevent NRG1-induced demyelination by enhancing the proteasomal degradation of c-Jun.This work was supported the Juvenile Diabetes Research Foundation and The National Institutes of Health [grant numbers NS054847 (to R.T.D.), CA120458 and CA109265 (to B.S.J.B.) and NS075311 (to B.S.J.B. and R.T.D.)]

Chromosome alterations and E-cadherin gene mutations in human lobular breast cancer

We have studied a set of 40 human lobular breast cancers for loss of heterozygosity (LOH) at various chromosome locations and for mutations in the coding region plus flanking intron sequences of the E-cadherin gene. We found a high frequency of LOH (100%, 31/31) at 16q21–q22.1. A significantly higher level of LOH was detected in ductal breast tumours at chromosome arms 1p, 3p, 9p, 11q, 13q and 18q compared to lobular breast tumours. Furthermore, we found a significant association between LOH at 16 q containing the E-cadherin locus and lobular histological type. Six different somatic mutations were detected in the E-cadherin gene, of which three were insertions, two deletions and one splice site mutation. Mutations were found in combination with LOH of the wild type E-cadherin locus and loss of or reduced E-cadherin expression detected by immunohistochemistry. The mutations described here have not previously been reported. We compared LOH at different chromosome regions with E-cadherin gene mutations and found a significant association between LOH at 13 q and E-cadherin gene mutations. A significant association was also detected between LOH at 13q and LOH at 7q and 11q. Moreover, we found a significant association between LOH at 3 p and high S phase, LOH at 9p and low ER and PgR content, LOH at 17p and aneuploidy. We conclude that LOH at 16q is the most frequent chromosome alteration and E-cadherin is a typical tumour suppressor gene in lobular breast cancer. © 1999 Cancer Research Campaig

Endothelial cell CD36 deficiency prevents normal angiogenesis and vascular repair

Endothelial cells (ECs) maintain vascular integrity and mediate vascular repair and angiogenesis, by which new blood vessels are formed from pre-existing blood vessels. Hyperglycemia has been shown to increase EC angiogenic potential. However, few studies have investigated effects of fatty acids (FAs) on EC angiogenesis. Cluster of differentiation 36 (CD36) is a FA transporter expressed by ECs, but its role in EC proliferation, migration, and angiogenesis is unknown. We sought to determine if circulating FAs regulate angiogenic function in a CD36-dependent manner. CD36-dependent effects of FAs on EC proliferation and migration of mouse heart ECs (MHECs) and lung ECs (MLECs) were studied. We used both silencing RNA and antisense oligonucleotides to reduce CD36 expression. Oleic acid (OA) did not affect EC proliferation, but significantly increased migration of ECs in wound healing experiments. CD36 knockdown prevented OA-induced increases in wound healing potential. In EC transwell migration experiments, OA increased recruitment and migration of ECs, an effect abolished by CD36 knockdown. Phospho-AMP-activated protein kinase (AMPK) increased in MHECs exposed to OA in a CD36-dependent manner. To test whethe

Novobiocin Analogs That Inhibit the MAPK Pathway

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of Medicinal Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/acs.jmedchem.5b01354.Heat shock protein 90 (Hsp90) inhibition by modulation of the N-or C-terminal binding site has become an attractive strategy for the development of anti-cancer chemotherapeutics. The first Hsp90 C-terminus inhibitor, novobiocin, manifested a relatively high IC50 value of ~700 μM. Therefore, investigation of the novobiocin scaffold has led to analogs with improved antiproliferative activity (nanomolar concentrations) against several cancer cell lines. During these studies, novobiocin analogs that do not inhibit Hsp90 were identified; however, these analogs demonstrated potent anti-proliferative activity. Compound 2, a novobiocin analog, was identified as a MAPK pathway signaling disruptor that lacked Hsp90 inhibitory activity. In addition, structural modifications of compound 2 were identified that segregated Hsp90 inhibition from MAPK signaling disruption. These studies indicate that compound 2 represents a novel scaffold for disruption of MAPK pathway signaling and may serve as a useful structure for the generation of new anti-cancer agents